Composition and method for monitoring lipid

ABSTRACT

A method of detecting a condition in a subject comprises the steps of contacting cells of the subject with single-walled carbon nanotubes (SWCNTs), monitoring photoluminescence emitted by SWCNTs internalized into the cells and generating an SW-CNT emission profile, comparing the SWCNT emission profile to a control emission profile for the SWCNTs to produce a result, and determining a likelihood of having the condition in the subject based on the result from the comparing step. Also disclosed is a method for screening agents capable of changing endocytic environment using SWCNTs.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application claims priority of U.S. Provisional Application No.62/004,122, filed on May 28, 2014. The entirety of the aforementionedapplication is incorporated herein by reference.

This invention was made with Government support under NationalInstitutes of Health Grants DP2-HD075698 and R37-DK27083. The Governmenthas certain rights in the invention.

FIELD

The present disclosure relates generally to devices and methods formonitoring lipid and, in particular, lipid in a biological sample orintracellular lipid using carbon nanotube-based optical reporters.

BACKGROUND

Lysosomes are vacuolar organelles responsible for the breakdown oflipids, proteins, sugars, and other cellular material into theirconstituent components. To degrade intracellular organelles, lysosomesfuse with autophagosomes to form autolysosomes, while extracellularcargo marked for further processing and degradation is directed to thelysosome from the endolysosomal pathway itself. In addition, lysosomesare involved in nutrient sensing of engulfed amino acids. Duringstarvation, mTORC1 is inhibited and autophagy is induced, thusindicating lysosomes as a link between nutrient availability andsignaling pathways related to cell growth.

A growing body of work implicates lysosomal dysfunction in a range ofpathologies. Dysfunction in the ability of lysosomes to catabolize orexport their contents results in lysosomal storage disorders, a familyof diseases characterized by pathologies caused by the accumulation ofundigested substrates. Lysosomal cholesterol accumulation is causalduring inflammation in both atherosclerosis and non-alcoholicsteatohepatitis. Defects in the fusion of lysosomes with autophagosomesleads to an accumulation of autolysosomes in the neurons of patientswith amyotrophic lateral sclerosis (ALS), while impaired autolysosomalproteolysis is implicated in both Alzheimer's and Parkinson's diseases.The ability to observe changes in the lipid content of lysosomes iscrucial to understanding the distinct roles played by the lysosomes insuch a variety of diseases. However, no one has developed and/or appliedimaging or any other method to observe endogenous cholesterol and/orother lipids in live cells and animals. It would be advantageous to doso for a number of reasons including, for example, to measure and detectcholesterol and other lipids in early stage disease detection.

Spectral imaging is a powerful tool for detection, validation,separation, and quantification in applications ranging from mineralassessment of geological satellite images to semiconductor materialcharacterization. In contrast to multi-spectral imaging in discretewavelength bands, hyper-spectral imaging produces a full,quasi-continuous emission spectrum at every spatial pixel. Severalmethods exist for hyperspectral data acquisition, includingpixel-by-pixel, line-by-line acquisition of spectra, or globally byacquiring separate images at each wavelength. Recent applications ofglobal hyperspectral imaging have used volume Bragg gratings (VBG) toacquire spectrally-defined images from the scanned wavelength space, forthe mapping of solar cell saturation currents and in astronomicalimaging.

SUMMARY

One aspect of the application relates to a method of detecting acondition in a subject using single-walled carbon nanotubes (SWCNTs).The method comprises the steps of contacting cells of a subject withSWCNTs, monitoring photoluminescence emitted by SWCNTs internalized intothe cells and generating an SWCNT emission profile, comparing the SWCNTemission profile to a control emission profile for the SWCNTs, anddetermining a likelihood of having the condition in the subject based ona result from the comparing step.

Another aspect of the application relates to a method for screeningagents capable of changing endocytic dielectric environment. The methodcomprises the steps of contacting testing cells with a candidate agent,wherein the testing cells contain internalized SWCNTs, monitoringphotoluminescence emitted by internalized SWCNTs and generating a SWCNTemission profile, and comparing the SWCNT emission profile to a controlemission profile for the SWCNTs to produce a result, wherein thecandidate agent is an agent that is capable of changing endocyticdielectric environment if the result shows a significant differencebetween the SWCNT emission profile to a control emission profile.

Another aspect of the application relates to a method for measuringlipid content in a sample. The method comprises the steps of mixing thesample with SWCNTs, monitoring photoluminescence emitted by the SWCNTsand generating a sample SWCNT emission profile, comparing the sampleSWCNT emission profile to a control SWCNT emission profile or emissionvalue, and determining a lipid content in the sample based on the resultof said comparing step.

In some embodiments, the SWCNTs are semi-conductive SWCNTs. In otherembodiments, the SWCNTs are polymer-coated non-covalently encapsulatedSWCNTs. In other embodiments, the SWCNTs are encapsulated by DNAoligonucleotides, wherein the DNA oligonucleotides are ss(GT)₆oligonucleotides. In other embodiments, the SWCNTs have chiral indices(n,m) of (6,5) or (8,6). In other embodiments, the SWCNTs are ss(GT)₆(8,6) SWCNTs.

In some embodiments, the SWCNT emission profile detected is asolvatochromatic response which indicates that photoliuminescenceemitted by SWCNTs is blue-shifted, wherein the blue-shifted emissionindicates substantial lipid (such as cholesterol) accumulation in thelocal dielectric environment.

In some embodiments, the condition is a disease linked to elevated lipidor cholesterol content. In other embodiments, the condition is alysosomal storage disorder selected from the group consisting ofNiemann-Pick, Tay-Sachs and Gaucher's disease. In another embodiment,the condition is hypercholesterolemia. In another embodiment, thecondition is a disease selected from the group consisting ofatherosclerosis, coronary heart diseases, stroke, diabetes, fatty liverdisease and cancer. In another embodiment, the cells contacted withSWCNTs are cultured in vitro. In another embodiment, the cells contactedwith SWCNTs are located in a subject in vivo.

In another embodiment, the cells contacted with SWCNTs are cellsextracted from the subject. The method further comprises the steps of:extracting cells from the subject; incubating the extracted cells withSWCNTs; and monitoring a wavelength-shifted solvatochromatic response ofthe SWCNTs. In some embodiments, the wavelength-shifted solvatochromaticresponse is a blue-shifted emission by the SWCNTs. In some embodiments,the cells are fibroblast cells. In other embodiments, the cells aremicrophages. In other embodiments, the cells are hepatocytes. In otherembodiments, the cells are cancer cells. In other embodiments, the cellsare lymphocytes or blood cells.

The SWCNTs function as a carbon nanotube optical reporter (CNOR) viasolvatochromic shifting of its emission in response to a change in theimmediate dielectric environment, allowing solvatochromic measurementswhich are free of artifacts induced by photobleaching, concentration,and movement of the emitter through the optical field. When applied tolive cells, the CNOR wavelength transiently and reversibly reflected thechanging dielectric environment of the maturing endosome. In someembodiments, the CNOR measures a drug-induced increase in endosomalcholesterol or lipid via an enhanced blue-shift and is capable ofidentifying the high free cholesterol or lipid content in human patientfibroblasts with the NPC1 mutation and benchmarked disease-statereversal on treatment with cyclodextrin. Applications of the CNOR usinghyperspectral imaging will allow new functional measurements of analytesin living systems for the study of disease, drug screening and clinicalapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of thepresent disclosure and, together with the written description, serve toexplain the principles of the exemplary embodiments of the presentdisclosure.

FIG. 1 illustrates various aspects of reporter response to lipidicmolecules. Panel A illustrates normalized absorption and emissionspectra of the ss(GT)₆-encapsulated (8,6) SWCNTs; Panel B illustratesresponse of the emission wavelength of surface-adsorbed ssGT₆-(8,6)SWCNTs to solvent dielectric (error bar is from triplicate technicalreplicates); Panel C illustrates emission wavelength of ssGT₆-(8,6)SWCNTs in solution as a function of added LDL concentration; Panel Dillustrates overly of transmitted light image and hyperspectral image ofthe reporter in RAW 264.7 cells incubated in LPDS, and in LPDS withAc-LDL and U18666A added (color legend maps to nanotube emission peak);Panel E illustrates histogram from the combined emission fromss(GT)₆-(8,6) SWCNTs under the two conditions previously listed; Panel Fillustrates MD simulation 5 image of ss(GT)₆-(8,6) SWCNTs in water, andin the presence of additional cholesterol molecules; Panel G illustrateswater density on the nanotube surface for ss(GT)₆-(8,6) SWCNTs, and inthe presence of added cholesterol.

FIG. 2 illustrates various aspects of an exemplary SWCNT solvatochromicresponse to endosomal dielectric environment. Panel A is an illustrationof exemplary time-course histograms of ss(GT)₆-encapsulated (8,6) SWCNTsemission from live HeLa cells showing that at 30 minutes, a narrowdistribution of emission wavelengths was observed, centered at1200.7±0.4 nm and after 6 hours of incubation, SWCNTs in the majority ofendosomes were blue-shifted, and 24 hours after incubation, a singleblue-shifted population remained; Panel B is an illustration ofexemplary center wavelengths of SWCNTs emission ROIs overlaid ontransmitted light images of the HeLa cells wherein a progressiveblue-shifting in individual endosomes was observed; Panel C is anillustration of exemplary microtubule polymerization inhibitornocodazole prevented blue-shifting, while the steroid U18666Aexacerbated blue-shifting of SWCNTs emission in HeLa cells relative tocontrol conditions (mean±SD, n=3 trials); Panel D is an illustration ofexemplary overlays of SWCNTs emission over brightfield images (top) andfilipin staining (bottom) for the same experimental conditions;

FIG. 3 illustrates various aspects of an exemplary live-cell reportingof Niemann-Pick C disease and its therapeutic reversal in lysosomalstorage organelles. Panel A is an illustration of an exemplary (8,6)nanotube in wild-type (WT) fibroblasts exhibited a narrow, red-shifteddistribution at the 24 hour time point showing nanotubes incubated inNPC fibroblasts reported both a red population and a broad, blue-shiftedpopulation; Panel B is an illustration of an exemplary nanotube emissionoverlaid on transmitted light images show the spatial distribution ofthis spectral heterogeneity; Panel C shows (a) the SWCNTs in WTfibroblasts maintained their red-shifted emission over 48 hours, (b)that after 48 hours in NPC fibroblasts, the SWCNTs emission became broadand blue-shifted, and (c) that after 24 hours of treatment with 100 μMHPβCD (48 hours after the experiment began), the SWCNTs population inNPC fibroblasts had red-shifted to near-WT levels; Panel D is anillustration of an exemplary nanotube/transmitted light overlays showingthe SWCNTs response and reversal quantitatively and spatially andshowing that filipin staining of fixed cells under similar conditionsconfirms the accumulation of cholesterol in NPC cells without HPβCD;Panel E is an illustration of an exemplary nanotube spectral differencesin WT, NPC, and cyclodextrin-treated NPC cells plotted with standarderror (mean±SD, n=3);

FIG. 4 illustrates various aspects of detection of lysosomal storagedisorders. Panel A illustrates mean nanotube emission from wild-typefibroblasts at 24 hours and 48 hours, from patient-derived NPC1fibroblasts at 24 hours and 48 hours, and from NPC1 fibroblasts treatedwith cyclodextrin for 24 hours, 24 hours after nanotube addition; PanelB illustrates histograms of the nanotube emission from single lysosomesat 48 hours, from wild-type fibroblasts, NPC1 and NPC1 cells treatedwith cyclodextrin for 24 hours; Panel C illustrates mean filipinintensity from WT fibroblasts, NPC1 and NPC1 cells treated withcyclodextrin for 24 hours, at 48 hours after nanotube addition; Panel Dillustrates mean emission from nanotubes in WT fibroblasts, NPC1, andNPC1 pretreated with cyclodextrin for 24 hours prior to nanotubeaddition; Panel E illustrates mean emission from nanotubes in RAW 264.7macrophages in DMEM+10% FBS media, in media with 3 ug/L U18666A, and inmedia with 10 uM Lalistat; Panel F illustrates the histogramscorresponding to Panel E; Panel G illustrates model for nanotubeemission shift in normal lysosomes, lysosomes with NPC1 phenotypeinduced with U18666A, and in lysosomes with Woiman's disease inducedwith Lalistat (all errors bars are S.E.M. from 3 triplicateexperiments).

FIG. 5 illustrates various aspects of single cell kinetics of lipidaccumulation. Panel A illustrates RAW macrophages in lipoproteindepleted serum (LPDS), incubated with nanotubes for 3 hours (Ac-LDL (100ug/mL) and U18666A (3 ug/mL) added at t=0 minutes); Panel B illustratesmean emission from 4 independent time courses and single celltrajectories of lysosomal lipid accumulation (error bars=standarddeviation, from 4 independent experiments); Panel C illustratesdistribution of time constants from single cells undergoing lysosomallipid accumulation, fit with a log-normal distribution; Panel Dillustrates a scatter plot of the starting nanotube emission wavelengthagainst the time constant for lipid accumulation for 60 single cells.

FIG. 6 illustrates various aspects of spatially and temporallycorrelating lysosomal lipid content to macrophage differentiation. PanelA illustrates changes in macrophage differentiation markers CD11b andF4/80, and the monocyte marker GR1+ on BMDM differentiating in thepresence of CSF1; Panel B illustrates transmitted light and fluorescentimage of nanotubes localized within the lysosomes of BMDM; Panel Cillustrates hyperspectral image of nanotubes in BMDM at days 3 and 5;Panel D illustrates histogram of the mean nanotube emission from BMDMcells at days 3 and 5; Panel E illustrates hyperspectral image of twoBMDM cells at day 3, and their corresponding normalized Simpson's Index;Panel F illustrates scatter plot of normalized Simpson's Index with themean emission from individual cells.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to use the present methods and kits. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present methods and kits. However, it will beapparent to one skilled in the art that these specific details are notrequired to practice the use of the methods and kits. Descriptions ofspecific applications are provided only as representative examples. Thepresent methods and kits are not intended to be limited to theembodiments shown, but are to be accorded the widest possible scopeconsistent with the principles and features disclosed herein.

Headings used herein are for organizational purposes only and are notmeant to be used to limit the scope of the description or the claims. Asused throughout this application, the word “may” is used in a permissivesense (i.e., meaning having the potential to), rather than the mandatorysense (i.e., meaning must). The terms “a” and “an” herein do not denotea limitation of quantity, but rather denote the presence of at least oneof the referenced items.

One aspect of the application relates to a method of detecting acondition in a subject using single-walled carbon nanotubes (SWCNTs).The method comprises the steps of contacting cells of a subject withSWCNTs, monitoring photoluminescence emitted by SWCNTs internalized intosaid cells and generating an SWCNT emission profile; comparing the SWCNTemission profile to a control emission profile for the SWCNTs; anddetermining a likelihood of having said condition in said subject basedon a result from said comparing step. In some embodiments,photoluminescence emitted by SWCNTs is monitored by hyperspectralimaging.

Nanotubes

As used herein, the term “single-walled carbon nanotubes (SWCNTs)”refers to allotropes of carbon with a cylindrical nanostructure. MostSWCNTs have a diameter of close to 1 nanometer, with a tube length thatcan be many millions of times longer. The structure of a SWCNT can beconceptualized by wrapping a one-atom-thick layer of graphite calledgraphene into a seamless cylinder. The way the graphene sheet is wrappedis represented by a pair of indices (n,m). The integers n and m denotethe number of unit vectors along two directions in the honeycomb crystallattice of graphene. If m=0, the nanotubes are called zigzag nanotubes,and if n=m, the nanotubes are called armchair nanotubes. Otherwise, theyare called chiral nanotubes. SWCNTs are an important variety of carbonnanotube because most of their properties change significantly with the(n,m) values, and this dependence is non-monotonic. In particular, theirband gap can vary from zero to about 2 eV and their electricalconductivity can show metallic or semiconducting behavior. For a given(n,m) nanotube, if n=m, the nanotube is typically metallic; if n−m is amultiple of 3, then the nanotube is typically semiconducting with a verysmall band gap, otherwise the nanotube is typically a moderatesemiconductor. Carbon nanotubes are available from a variety ofmanufacturers, and may be obtained as sets of unpurified nanotubeswithin which there are a variety of distinct species present.

In some embodiments, the SWCNTs are semiconducting SWCNTs.Semiconducting SWCNTs exhibit intrinsic photoluminescence which isnarrow-band and uniquely photostable. The intrinsic photoluminescence ofsemiconducting SWCNTs are sensitive to changes in the local dielectricenvironment, which may result in intensity modulation as well aswavelength shifting (i.e. solvatochromism) of the photoluminescenceprofile of the semiconducting SWCNTs. The instant application utilizesspatial measurement of the spectral diversity and response ofsemiconducting SWCNTs in complex biological environments to determinethe presence or absence of certain diseased conditions. In someembodiments, the SWCNTs have chiral indices of (6,5) or (8,6).

The SWCNTs may be a mixture of SWCNTs with different chiral indices. Insome embodiments, the SWCNTs are a mixture of 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more SWCNT species of different chiralindices. In other embodiments, purified or partially purified SWCNTspecies is used. In some embodiments, the SWCNTs have chiral indices of(8,6) or (6,5).

In some embodiments, the SWCNTs of the present application have anaverage diameter of 0.1-2 nm, 0.3-1.6 nm or 0.6-1.3 nm. In someembodiments, the SWCNTs of the present application produce fluorescencewith a wavelength between about 400-1800 nm, 600-1600 nm, 900-1500 nm,or 1000-1200 nm in an appropriate endocytic dielectric environment. Insome embodiments, fluorescence within the claimed ranges is producedusing excitation light in the wavelength range of 700-900 nm. In someembodiments, the excitation light has a wavelength of about 730 nm. isdetected with wavelength between about 730-880 nm. In some embodiments,a specialized detector is used for fluorescent light with wavelengthabove 1050 nm.

Nanotubes are not water-soluble, and need to be solubilized or suspendedto facilitate intake by cells. In some embodiments, SWCNTs areencapsulated with oligonucleotides (such as single stranded DNA) orsynthetic polymers (such as polycarbodiimide or other amphiphilicpolymers). Lipids and surfactants may all be used to create a stablesuspension of nanotubes. Solubilization/suspension of nanotubes occursby mixing the chosen encapsulating agent, such as an oligonucleotide ora synthetic polymer, with the nanotube in a weight ratio ranging from1:1 to 4:1, followed with sonication. Sonication may be performed in avariety of ways, including probe tip ultrasonication and the milder bathsonication.

A large range of DNA oligonucleotides, both in terms of length andsequence, can be used to stably suspend nanotubes. In some embodiments,the oligo nucleotides are single stranded (ss) oligo nucleotides with alength of 2-90 nucleotides. In some embodiments, the oligonucleotideshave sequences of ss(TAT)_(n), ss(AC)_(n), ss(ACT)_(n) and ss(GT)_(n),with n=2-40. In some embodiments, the oligos are ss(GT)₆, ss(GT)₁₅,ss(ACT)₆ or ss(TAT)₄, ss(TAT)₁₅. In some embodiments, the oligonucleotides are double stranded oligo nucleotides. In other embodiments,SWCNTs are suspended in detergents, such as sodium deoxycholate (SDC),sodium cholate (SC), sodium dodecylbenzene sulfonate (SDBS) or sodiumdodecyl sulfate (SDS). In some embodiments, SWCNTs are encapsulated witholigonucleotides by sonication with oligonucleotides in a buffersolution, such as phosphate buffered saline (PBS). In some embodiments,the SWCNTs are ss(GT)₆ encapsulated (8,6) SWCNTs (also referred to as“ss(GT)₆-(8,6) nanotubes”) or ss(GT)₆ encapsulated (6,5) SWCNTs (alsoreferred to as “ss(GT)₆-(6,5) nanotubes”).

Certain nanotube species are better sensors for lipids than others, Oneof ordinary skill in the art will understand that the present inventionis not limited to any one specific chirality. Nanotubes that areencapsulated with short DNA sequences are preferred for detection oflipids within a particular biological environment. One of ordinary skillwill understand that the (8,6) nanotube has been chosen simply as annon-limiting example. There is no limitation upon the present inventionof the nature or complexity of the purification technique used to obtaina particular nanotube species, and any technique which may enableindependent control of DNA sequence (such as synthetic biologyapproaches) and or control of nanotube chirality may be used. There isalso no limitation upon the present invention to only pure samples of aparticular nanotube species, the present invention may use either mixedor pure samples of polymer-encapsulated carbon nanotubes at thediscretion of researchers employing the disclosed technique.

Disease Conditions

The compositions, devices and methods of the present application may beused for the detection of disease conditions such as atherosclerosis,coronary heart diseases, stroke, diabetes, cancer, lysosomal storagedisorders, hypercholesterolemia and other diseases linked to elevatedlipid and cholesterol content.

The term “lysosomal storage disorder” may refer to any of a group ofdiseases resulting from abnormal metabolism resulting in accumulation ofa substrate, such as glycolipids, in the lysosome. Lysosomal storagedisorders are caused by lysosomal dysfunction usually as a consequenceof deficiency of a single enzyme required for the metabolism of lipids,glycoproteins or mucopolysaccharides. These diseases include, but arenot limited to the following: Pompe Disease, Hurler syndrome(Mucopolysaccharidosis type I; MPS-1), Hunter syndrome (MPS-II),Sanfilippo syndrome A (MPS-IIIA), Sanfilippo syndrome B (MPS-IIIB),Sanfilippo syndrome C (MPS-IIIC), Sanfilippo syndrome D (MPS-IIID),classic Morquio syndrome (MPS-IVA), Morquio B Disease (MPS-IVB),Maroteaux-Lamy syndrome (MPS-VI), Sly syndrome (MPS-VII), Sialidosis(Mucolipidosis type I; ML-I), I-cell Disease (ML-II), Pseudo-HurlerPolydystrophy (ML-III), Schindler Disease/Kanzaki Disease,α-Mannosidosis, β-Mannosidosis, α-Fucosidosis, Aspartylglucosaminuria,Niemann-Pick Disease Type C, Niemann-Pick Disease Type D, Neuronalceroid lipofuscinoses, Wolman Disease, Acid lipase disease, Fabry'sDisease, Niemann-Pick-Disease Type A, Niemann-Pick Disease Type B,Gaucher's Disease, Krabbe's Disease, GM1 Gangliosidosis, Tay-SachsDisease, Sandhoff Disease, Metachromatic Leukodystrophy, Farber Disease,Multiple suifatase deficiency, and Galactosialidosis.

As used herein the term “cancer” refers to any of the various malignantneoplasms characterized by the proliferation of cells that have thecapability to invade surrounding tissue and/or metastasize to newcolonization sites, including but not limited to carcinomas, sarcomas,melanoma and germ cell tumors. Exemplary cancers include bladder cancer,brain cancer, breast cancer, ovarian cancer, cervix cancer, coloncancer, head and neck cancer, kidney cancer, lung cancer, mesothelioma,prostate cancer, stomach cancer and uterus cancer.

As used herein the term “hypercholesterolemia” refers to the presence ofhigh levels of cholesterol in the blood. Cholesterol is a sterol whichis precursor of the steroid hormones, bile acids and vitamin D. Elevatedlevels of LDL-cholesterol is associated with an increased risk ofatherosclerosis and coronary heart disease.

As used herein the term “hyperspectral imaging” is a method of imagingspectroscopy that generates a map of a region of interest based on localchemical composition. In hyperspectral imaging, a two-dimensional imageis created having a spectral data inherent in each pixel. These stacksof images comprise a “hypercube.” It is possible to correlate thespectrum of each pixel with the presence and concentration of variouschemical species. This data can then be construed as a “gradient map” ofthese species in a surface.

A “subject” refers to either a human or non-human animal. Examples ofnon-human animals include vertebrates, e.g., mammals, such as non-humanprimates (particularly higher primates), dogs, rodents (e.g., mice,rats, or guinea pigs), pigs and cats, etc. In a preferred embodiment,the subject is a human. A “biological sample” may refer to a tissue,cell, blood or plasma sample from a subject.

Another aspect of the application relates to a method of monitoring thelocal dielectic environment in a tissue culture using SWCNTs. The methodcomprises the steps of contacting cells of a tissue culture of a subjectwith SWCNTs, monitoring photoluminescence emitted by SWCNTs internalizedinto said cells and generating an SWCNT emission profile; comparing theSWCNT emission profile to a control emission profile for the SWCNTs; anddetermining that a change in the local dielectric environment hasoccurred within the cells of the tissue culture because a blue-shift orred-shift is observed in the wavelength of light reflected by theSWCNTs.

As used herein, the term “blue-shift” refers to a decrease inwavelength, with a corresponding increase in frequency, ofelectromagnetic waves. In visible light, this shifts the color from thered end of the spectrum to the blue end. The term also applies whenphotons outside the visible spectrum (e.g., X rays and radio waves) areshifted toward shorter wavelengths, as well as to shifts in the deBroglie wavelength of particles. In some embodiments, a “blue-shift” isdefined as a decrease of average wavelength or median wavelength of atleast 0.01, 0.03, 0.05, 0.07, 0.09, 0.1, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20, 30, 40, 50 nm.

As used herein, the term “red-shift” refers to an increase inwavelength, with a corresponding decrease in frequency, ofelectromagnetic waves. In visible light, this shifts the color from theblue end of the spectrum to the red end. The term also applies whenphotons outside the visible spectrum (e.g., X rays and radio waves) areshifted toward longer wavelengths, as well as to shifts in the deBroglie wavelength of particles. In some embodiments, a “red-shift” isdefined as an increase of average wavelength or median wavelength of atleast 0.01, 0.03, 0.05, 0.07, 0.09, 0.1, 0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20, 30, 40, 50 nm

Control Emission Profile

The control emission profile is the emission profile of a given SWCNT oran oligo-encapsulated SWCNT at a predetermined condition. In someembodiments, the control emission profile is the emission profile of aninternalized SWCNT or a plurality of internalized SWCNTs in a normalcell or in a cell that is free from a given condition. In otherembodiments, the control emission profile is a emission profile obtainedat an earlier time point (e.g., prior to exposure to an test agent, orprior to the development of a condition, or prior to the initiation of atreatment regimen) in the same cell or cells.

Method of Detection of A Lysosomal Storage Disorder

In some embodiments, the present disclosure pertains to a method fordetecting lysosomal storage disorder by measuring the endocyticdielectric environment in fibroblasts obtained from a subject usingSWCNTs encapsulated in a specific short oligonucleotide. A decrease inthe dielectric constant of the endosomal environmental was detected viaa distinct solvatochromic blue-shift in emission. A spectrometer,fluorometer, plate reader apparatus, or combinations thereof may beemployed for the detection of solvatochromic shift. In one specificembodiment, a spectral imaging/hyperspectral microscope is employed.

In one embodiment, the invention is a method of detection of a lysosomalstorage disorder comprising: contacting a tissue, cell, blood or plasmasample from a subject with SWCNTs, determining the fluorescence profileof the SWCNTs and comparing the profile to a control profile of theSWCNTs. If exposure to the tissue, cell, blood or plasma results in asolvatochromic blue-shift of the fluorescence profile of the SWCNTs fromthe control profile, the subject is at risk of the lysosomal storagedisorder.

SWCNTs when used as CNORs undergo a shift in their emission wavelength.In particular, when an SWCNT undergoes a shift in emission wavelengthwhen it interacts with lipids in close proximity, including cholesteroland lipoproteins. When in solution, the SWCNTs of the present inventioncan detect LDL and total lipid content in solution, whole blood, andpatient serum with high sensitivity. When on a surface (e.g. immobilizedon a surface as part of a device), the SWCNTs of the present inventioncan detect LDL and total lipid content at low concentrations. When incells, the SWCNTs can detect lipid accumulation in the endosomes andlysosomes of live cells. In particular, SWCNTs can detect the directincrease in lipid accumulation in lysosomal storage disorders, such asNiemann Pick Type C or Wolman's Disease. The SWCNTs of the presentinvention can be used as a screening tool in live cells obtained frompatients who have cells with lipid accumulation. This provides analternative to techniques such as filipin staining used for NPC1.

Lysosomal lipid dysfunction, is correlated with a variety of diseases.For example, non-alcoholic fatty liver disorder (NAFLD, NASH, steatosis)has been shown to cause lysosomal lipid dysfunction in hepatocytes,kupffer cells, and stellate cells from human patients. In certainneurological disorders lipid accumulation in the lysosomes is implicateddue to autophagy increasing lipid content in the lysosomes, and has beendirectly implicated in a variety of diseases. In certain instances,onset of atherosclerosis is associated with lipid droplet formation,which occurs via the lysosomal pathway. Accordingly, all these diseasesor any other disease that is caused by or correlated with lysosomallipid accumulation may be detectable by the methods disclosed herein.

Method of Screening for Drugs To Treat Lysosomal Storage Disorders

In another aspect, the instant application utilizes spatial measurementof the spectral diversity and response of semiconducting SWCNTs incomplex biological environments to determine the effect of an agent onthe local dielectric environment of a cell.

In one embodiment, a method of screening for drugs to treat lysosomalstorage disorders comprises the steps of introducing semiconductingSWCNTs into cells obtained from a subject with a lysosomal storagedisorder, determining the fluorescence profile of the SWCNTs, exposingthe cells to a candidate agent, determining the fluorescence profile ofthe SWCNTs after exposure to the agent, and identifying the agent as acandidate for treatment of lysosomal storage disorders when the exposureto the agent results in a red-shift of the fluorescence profile of theSWCNTs.

In another embodiment, a method of analyzing the efficacy of treatmentof lysosomal storage disorders comprises the steps of introducingsemiconducting SWCNTs into cells obtained from a subject with alysosomal storage disorder at different stages of treatement,determining the fluorescence profile of internalized SWCNTs at differentstages of treatment, wherein a red-shift of the fluorescence profile ofthe SWCNTs during the course of treatment indicates efficacy of thetreatment. In some embodiments, the semiconducting SWCNTs are implantedin vivo in the subject and the fluorescence profile of internalizedSWCNTs at different stages of treatment is determined in vivo.

The nanotube emissions from solutions or cells in a 96/384 well plateformat may be obtained by an appropriately designed instrument. Such aninstrument can be used for high throughput screening for drugs thatincrease or decrease lipid accumulation in the lysosomes. For example,the present invention may be used for detection and screening forphospholipidosis, a condition characterized by cholesterol accumulationin the lysosomes, which is a side effect in human cells and induced by avariety of drugs currently in human use.

Nanotube emissions are also detectable in small animal in vivo modelsvia appropriately designed pre-clinical instruments. In vivo models fordisease progression may be assayed via lysosomal lipid accumulationtracked by the techniques disclosed herein. Disease onset for a varietyof disorders as discussed herein, and methods of intervention to treatsuch diseases can be assayed by acquiring the nanotube emissions fromanimal models over a period of time using the techniques disclosedherein.

The particular design of the instruments for detection of nanotubeemissions as disclosed herein is not limiting on the present invention.For example, a hand-held probe, such as a wand-like device, has beenused to detect nanotube emissions (and related shifts in emissionwavelengths) in animal models and may be used in human patients.Nanotube emission in the near-infra red is preferred for detectionthrough human tissue than emission at other wavelengths.

Kits

Another aspects of the instant application relates to a kit fordetecting endocytic dielectric environment. In some embodiments, the kitcomprises SWCNTs and oligo nucleotides in an amount sufficient toencapsulate the single-walled carbon nanotubes. In some embodiments, thekit contains SWCNTs encapsulated in oligo nucleotides. In someembodiments, the kit contains ss(GT)₆-(8,6) nanotubes.

Another aspect of the instant application relates to oligonucleotideencapsulated SWCNTs. In some embodiments, the oligonucleotideencapsulated SWCNTs are selected from the groups consisting ofss(GT)₆-(8,6) SWCNTs, ss(GT)₆-(6,5) SWCNTs, ss(GT)₁₅-(8,6) SWCNTs,ss(GT)₁₅-(6,5) SWCNTs, ss(ACT)₆-(8,6) SWCNTs, ss(ACT)₆-(6,5) SWCNTs,ss(TAT)₄-(8,6) SWCNTs, ss(TAT)₄-(6,5) SWCNTs, ss(TAT)₁₅-(8,6) SWCNTs andss(TAT)₁₅-(6,5) SWCNTs. In other embodiments, the oligonucleotideencapsulated SWCNTs are ss(TAT)_(n−)-(8,6) SWCNTs, ss(AC)_(n−)-(8,6)SWCNTs, ss(ACT)_(n)-(8,6) SWCNTs, ss(GT)_(n−)-(8,6) SWCNTs,ss(TAT)_(n−)-(6,5) SWCNTs, ss(AC)_(n−)-(6,5) SWCNTs, ss(ACT)_(n)-(6,5)SWCNTs, ss(GT)_(n−)-(6,5) SWCNTs, wherein n is an integer between 2-40.

The description herein is for the purpose of teaching the person ofordinary skill in the art how to practice the present disclosure, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. The specific embodiments of the present application havebeen presented for purposes of illustration and description. They arenot intended to be exhaustive or to limit the application and method ofuse to the precise forms disclosed. Obviously many modifications andvariations are possible in light of the above teaching. It is understoodthat various omissions or substitutions of equivalents are contemplatedas circumstance may suggest or render expedient, but is intended tocover the application or implementation without departing from thespirit or scope of the claims of the present application.

EXAMPLES Example 1: Materials and Methods

The below materials and methods were used in the examples herein.

Hyperspectral Microscope System Excitation: A continuous wave (CW) 730nm diode laser with an output power of 1 W was injected into a multimodefiber to produce the excitation source for photoluminescenceexperiments. To ensure a homogenous illumination over the entiremicroscope field of view, the excitation beam passed through a custombeam shaping module to produce a top hat intensity profile within 20%variation on the surface of the sample under test. A long pass dichroicmirror with a cut-on wavelength of 880 nm was aligned to reflect thelaser into an Olympus IX-71 inverted microscope equipped with a 100X(UAPON100XOTIRF, NA=1.49) oil objective (Olympus, USA).

Hyperspectral Filter Construction: The hyperspectral filter was situatedin the system between the long-pass dichroic mirror and the camera. Todecrease the amount of photons produced by elastic laser scattering atthe surface of the sample, a long-pass filter with cut-on wavelength of815 nm was placed at the filter optical input. To ensure that the beamentirely passed through the volume Bragg grating (VBG) for filtering,the pupil of the optical system was imaged between two VBG passes by thefirst tube lens. Non-resonant light passes through a VBG un-diffracted.Only the wavelength that complies with the Bragg condition will resonatewith the grating and be diffracted in the direction of the corner cube.The corner cube was situated to reflect the filtered beam onto the VBGfor a second pass in order to cancel chromatic dispersion induced by thefirst pass and to narrow filtered bandwidth to 3.7 nm. The second tubelens formed a spectrally-filtered image on the IR camera sensor.

To acquire the spectrally filtered image at different wavelengths, thecorner cube and the VBG were positioned on rotation stages tocontinuously tune the diffracted wavelength. An automatic wavelengthcalibration process was designed to guarantee the relation between theangle of the rotation stage and the absolute wavelength of the grating.Using a xenon spectral lamp (Newport 6033), absolute wavelength accuracyof 0.5 nm was obtained.

Hyperspectral Microscope System Camera: A near-infrared hyperspectralfluorescence microscope was constructed by incorporating a volume Bragggrating to spectrally resolve the entire emitted image. A deep-cooledshort-wave infrared (SWIR) camera was designed for the hyperspectralsystem. By scanning the turret-mounted grating with respect to thecollimated emission, a continuous stack of 3.7 nm bandwidth images wasconstructed by a 2D InGaAs camera. A 2D InGaAs sensor array (nIRcamera), operational between 900 nm and 1700 nm and with a quantumefficiency superior to 70%, was used. The array, consisting of 320×256pixels with 30 μm pitch, was coupled with a four-stage TE cooler to keepthe sensor operating temperature below 190 K. The full-well capacity ofthe camera was 168000 electrons in high gain and 3.5 million electronsin low gain with 65 dB S/N ratio and 346 frames per second capability(full frame). The dynamic range was 14 bits and the readout noise was 57electrons at 346 fps in high gain. Images in the visible (400-700 nm)range were acquired using a QIClick digital CCD camera (Qimaging,Surrey, BC, Canada) attached to a separate port of the microscope.Hyperspectral cubes were acquired from 900-1400 nm for each of the256×320 pixels, thus providing 81,920 spectra per acquisition.

Hyperspectral Cube Rectification: Each point source produced acollimated beam having a different incident angle on the VBG, as animage is a sum of point sources issued from different positions of theobject seen by the microscope objective. Therefore, the angularselectivity of the grating resulted in a gradient in wavelength acrossthe field of view in the dimension parallel to the dispersion axis. Thefiltered image produced on the InGaAs camera was composed of a series ofvertical lines each with a specific wavelength. To obtain amonochromatic image, several frames at contiguous wavelengths must bescanned through in order to retrieve the wavelength of interest for eachimage. The reconstruction was performed using cubic interpolation onevery pixel for each monochromatic image according to the wavelengthcalibration parameters.

Nanotube Sample Preparation: Standard chemical reagents were purchasedfrom Sigma-Aldrich (St.Louis, Mo., US) and Fisher Scientific(Pittsburgh, Pa., US). Single-walled carbon nanotubes used throughoutthe study were commercially purchased and produced by the HiPco process(Unidym, Sunnyvale, Calif., US). Aqueous dispersions were created by theprobe-tip ultrasonication of (Sonics & Materials, Inc.) 2 mg of thespecified oligonucleotide (IDT DNA, Coralville, Iowa) with 1 mg of rawSWCNT in 1 mL of PBS (Gibco) for 30 minutes or 10 seconds at 40% of themaximum amplitude. Following ultrasonication, the dispersions wereultracentrifuged (Sorvall Discovery 90SE) for 30 minutes at 280,000×g.The top ¾ of the resultant supernatant was collected and itsconcentration was determined with a UV/Vis/nIR spectrophotometer (Jasco,Tokyo, Japan) using the extinction coefficient A₉₁₀=0.02554 L·mg⁻¹·cm⁻¹.To remove free DNA, 100 kDa Amicon centrithge filters (Millipore) wereused to concentrate and re-suspend the DNA-nanotube complexes.

To obtain near-pure (6,5) or (8,6)-nanotubes, HiPco sample was dispersedwith either ss(TAT)₄ or ss(GT)₆ oligonucleotides, respectively, andpurified according to a previously documented procedure (see X. M. Tu,S. Manohar, A. Jagota, M. Zheng, DNA sequence motifs forstructure-specific recognition and separation of carbon nanotubes.Nature 460, 250 (Jul. 9, 2009). Briefly, 1 mL of DNA-nanotube solution,after sonication and ultracentrifugation, was fed into a HydrocellCNT-NS1500 anion-exchange column (Biochrom) and eluted with a linearlyincreasing salt gradient of NaSCN using an HPLC (Agilent Technologies,CA, US). Sample purity was confirmed with absorption and estimated at90% (6,5) and 70% for (8,6).

Cell Lines and Cell Culture Procedures: HeLa CCL-2 cells (ATCC,Manassas, Va., US) were grown under standard incubation conditions at37° C. and 5% CO₂ in sterile-filtered DMEM with 10% heat-inactivatedFBS, 2.5% HEPES, 1% Glutamine, and 1% Penicillin/Streptomycin (allGibco). For studies performed with homozygous mutant NPC, compoundmutant heterozygote NPC, or wild-type fibroblasts, the cell linesGM18453, GM03123 or GM05659 (Coriell, Camden, N.J., US), respectively,were cultured in MEM with 10% FBS, 2.5% HEPES, and 1% Glutamine. Cellswere plated on glass-bottom petri dishes, or lysine-covered glass dishes(MatTek) for fibroblasts. Unpurified SWCNT were incubated at 1 mg/L andseparated SWCNT were incubated at 0.25 mg/L in media with cells for 30minutes at 37° C. Depending on the experiment, cells were imagedimmediately, or trypsinized (Gibco) and re-plated on a freshglass-bottom petri dish followed by hyperspectral imaging.

Experimental Data Acquisition: For glass surface-based measurements,DNA-nanotubes at 1 mg/L, were incubated on a glass-bottom 35 mm petridish (Mattek) for 10 seconds and immediately withdrawn by pipette. Thesurface was washed with PBS and fluorescence data were taken with 2 mLof fresh PBS covering the surface. While focusing on the surface, theindividual SWCNTs were excited with a 730 nm laser, ˜350 mW power at thesample, and wavelength data were taken from 900 to 1400 nm with 4 nmstep sizes and 4 s exposure time. Hyperspectral data from blank surfaceswere also taken to be used post-processing in background subtraction.

Data from hyperspectral cubes were analyzed by manually selectingfluorescent nanotube spots with 3×3 pixel ROIs. The mean intensity of aregion devoid of ROIs was defined as the background signal. Subtractingthe background value from each ROI resulted in an approximately zerobaseline for the spectrum from each nanotube.

Pharmacological treatments on HeLa cells were performed with Nocodazole(Sigma, 10 μg/mL added after SWCNT incubation) or U18666A (Sigma, 3μg/mL added 6 hours prior to SWCNT incubation). Fibroblast cells wereincubated with hydroxypropyl-β-cyclodextrin (Sigma, 100 μM) 6 hoursprior to SWCNT incubation.

Filipin staining was conducted by fixing cells with 1.5%paraformaldehyde in PBS for 20 minutes. Cells were then labeled with 50μg/mL filipin for 45 minutes. After washing, filipin images wereacquired using 350/50 nm excitation and 460/50 mm emission filters witha 400 nm dichroic long-pass filter (Olympus).

AFM imaging was conducted using freshly cleaved mica (Pelco Mica Disc,V1, Ted Pella) treated with an aqueous MgCl₂ (0.5 M) solution for 30seconds. Excess solution was removed and 10 μL aqueous suspension ofpurified ss(GT)₆-SWCNT (˜5 mg/L) was deposited onto the mica and allowedto stand for 20 seconds. The mica surface was then rinsed with water twotimes to remove unbound carbon nanotubes. The mica was then dried underultrapure nitrogen stream prior to AFM imaging. AFM images werecollected using an Asylum MFD-3D-BIO in AC mode using AC240TS tips(Asylum Research). The typical scan size was 2-5 μm; scan lines andpoints were 512, and the scan rate was 0.5 Hz-1.95 Hz.

Analysis Software: Hyperspectral data and visible data acquisition, aswell as cube rectification, were performed using the PhySpec software(Photon etc, Montreal, Canada) that also controls the hyperspectralmicroscope. Image processing and (region of interest) ROI selection wereconducted using ImageJ and FIJI using the Time Series Analyzer pluginand custom macros. Data analysis, curve fitting, and simulation programswere written in Matlab 2011 (The MathWorks, MA, US). Statisticalanalysis and graphs were generated using OriginPro 8.6 (OriginLab,Northampton, Mass.). AFM images were acquired and processed in IgorPro(Wavemetrics, OR, US).

Sodium deoxycholate (SDC)-suspended HiPCO SWCNTs adsorbed onto a glasssurface, were imaged in 0.1% SDC. After fitting each nanotube spectrumwith a Voigt function, fits with an R² value of less than 0.8 wererejected from all following analyses. The wavelength emission peak valuewas divided by the standard deviation of the baseline (not including theemission peak waveform itself) to obtain the SNR for the particularnanotube. A distribution of the SNR for the sample was created toextract statistical parameters.

Sorting the nanotube emission peaks in ascending wavelength orderseparated individual nanotube species, either by obvious gaps in thewavelength axis or via a k-means clustering algorithm. The exerciseresolved 14 (n,m) nanotube species and provided an absolute count of thenanotube population. Separated populations were assigned (n,m) valuesbased on an empirical Kataura plot as follows:

# (n, m) λstart λend 1 (8, 3) 960 970 2 (6, 5) 984 993 3 (7, 5) 10281038 4  (11, 0) 1042 1054 5  (10, 2) 1059 1076 6 (9, 4) 1107 1116 7 (8,4) 1117 1125 8 (7, 6) 1129 1145 9  (12, 1) 1179 1184 10 (8, 6) 1188 119511  (11, 3) 1206 1216 12  (10, 3) 1270 1275 13  (10, 5) 1276 1282 14 (8,7) 1284 1300

The SWCNTs, adsorbed to a glass substrate, was imaged in the presence ofDMEM+10% FBS (D10) before 0.1% (sodium deoxycholate) SDC was introducedto the buffer. The mean wavelength of the SWCNTs was 1201.5 nm.Hyperspectral cubes obtained after 10 minutes showed the SWCNTs emissionto reach 1190.5 nm. The shifting was reversed to 1199.1 nm on rinsingthe surface with water to remove SDC.

As these nanotubes responded to lipoproteins in solution and on asurface, they could be used to detect lipid accumulation in live cells.Nanotubes encapsulated with either ss(GT)₆ or ss(GT)₁₅ sequences wereincubated in media with HeLa cells at 1 mg/L for 30 minutes at 37° C.before being washed and imaged in fresh media. The spectra were analyzedto obtain the peak emission wavelengths of five nanotube species atdifferent time points. Data was reported as a change from the bulkwavelength value (nm) versus time after initial incubation.

Cells were incubated for 30 minutes with 0.25 mg/L of the SWCNTs beforerinsing and replacing with fresh media. Hyperspectral imaging wasconducted immediately afterwards and 24 hours later.

Nanotubes with fluorophore-labeled Cy3-ss(GT)₆ DNA were encapsulated andco-localized with the Cy3 emission with lysotracker to determine thatnanotubes remained localized within the endolysosomal pathway. It wasconfirmed that visible Cy3 emission also remains co-localized with thenIR emission from the nanotubes themselves. At a 2 pM incubationconcentration, no effect of the nanotubes on the proliferation orviability of the cells was observed.

Brightfield images of HeLa cells, and fluorescence images of LysoTrackerdye (Deep Red LysoTracker (ex/em 647/668 nm)), were obtained using aQIMAGING CCD camera attached to the hyperspectral microscope. Images ofnanotube emission in the same cells were obtained using the InGaAscamera on the same microscope. The two cameras had different aspectratios and pixel sizes, which were scaled to the same final image sizeand cropped to represent the same sample area. Emission from the SWCNT,(which shows as blue), was localized within the same regions as theemission from Lysotracker (which shows as red).

HeLa cells were incubated with purified (8,6) nanotubes for 30 minutes.Cells were then trypsinized and re-plated onto glass-bottom Petridishes. A second incubation was performed with (6,5) nanotubes for 30minutes (24 hours after the initial incubation). The live cells werethen imaged over the 900-1400 nm range with the hyperspectralmicroscope.

In a similar procedure to the experiment with homozygous NPC cells,fibroblasts with the compound heterozygous mutation of the NPC1 genewere plated on a poly-d-lysine-coated glass-bottomed petri-dish andimaged at different time points after an initial 30 minute incubationwith the SWCNTs. Histograms of the wavelength distribution profile ofthe SWCNTs were obtained.

Example 2: Analysis of Nanotube Emission

SWCNTs were non-covalently encapsulated in a single strandedoligonucleotide sequence to generate water soluble DNA-nanotubecomplexes. To test the hypothesis that an optimally short DNA sequencecould partially encapsulate the nanotube while leaving enough exposednanotube surface for hydrophobic molecules to bind to a 12 base longoligonucleotide, GT₆, was selected. GT₆ also functions as therecognition sequence for the (8,6) chirality. On encapsulating nanotubeswith GT₆, a mixture of GT₆-nanotubes composed of over 15 fluorescentnanotube chiralities were formed. Ion exchange chromatography wasperformed on this mixture to extract only the (8,6) chirality, soobtaining a pure GT₆-(8,6) sample. This GT₆-(8,6) sample was free ofmetallic and carbonaceous impurities, and consisted of a singleabsorption peak at 730 nm and an emission peak at 1200 nm (FIG. 1, panelA).

Surface adsorbed ss(GT)₆-(8,6) nanotubes were dried with ultrapure N₂ toremove any associated solution from the nanotube surface. Hyperspectralimages from single nanotubes in different solvent environments wereacquired to obtain the mean nanotube emission value for each solvent(FIG. 1, panel B). The emission wavelength of ss(GT)₆-(8,6) correlatedwell with the solvent dielectric (Spearman correlation of 0.89,p<0.01)—indicating that as the dielectric value of the environmentaround the nanotube decreases, the nanotube emission blue shifts.

The ss(GT)₆-encapsulated (8,6) nanotubes were introduced to a solutionof low density lipoprotein (LDL) to determine if their emissionwavelengths respond to whole lipoprotein. The emission shiftedmonotonically with LDL concentration in solution (FIG. 1, panel C),indicating that the nanotubes were sensitive to this lipid-richbio-assembly. The response of single nanotubes on a surface to lipidswas characterized and the detection of lipidic molecules by the nanotubewas found to be immediate and reversible.

Single-walled carbon nanotubes (HiPco), suspended with surfactant andadsorbed onto a glass substrate, were imaged under 730 nm excitation. Ahyperspectral cube of nanotube photoluminescence was acquired,spectrally resolving 14 distinct species indicated by their uniquechiral indices (n,m). Spectra of individual nanotubes displayed a highsignal to noise ratio (average 41.71) and were fit as Voigt profiles toobtain each nanotube's center wavelength, full width at half maximum(FWHM) and intensity. A population analysis of 238 individual nanotubes,using a k-means clustering algorithm, demarcated nanotube species suchthat their approximate quantities resembled those in the bulk HiPcosample. Analysis of the nanotube emission showed that the FWHM of allspecies in the 950-1350 nm range fell between 18-23 nm and exhibited aknown positive correlation with nanotube wavelength (Pearson'scorrelation=0.84 with p<0.005). Spectra from DNA-encapsulated (6,5)nanotubes (N=273), separated by ion exchange chromatography, wereobtained to determine the intrinsic variability in peak emissionwavelength and distribution of FWHMs for a single nanotube (n,m)species. The wavelength distribution centered at 990.22±0.35 nm with anaverage FWHM of 21.54±0.28 nm.

Example 3: Analysis of Nanotube Localization

Hyperspectral imaging of live HeLa cells incubated with nanotubesresolved 14 distinct internalized (n,m) species. HeLa cells wereincubated with 1 mg/L DNA-encapsulated HiPco for 10 minutes and unboundnanotubes were washed away prior to imaging. In agreement with previousstudies, the DNA-dispersed nanotubes entered cells via endocytosis andremained in late endosomes and lysosomes. After 30 minutes ofincubation, the cells were found to average one photoluminescentnanotube per endosome, measured by counting the number ofspatially-resolved emission bands per endosome. Two separated (n,m)nanotube species, were used to label sub-cellular structures.DNA-encapsulated (6,5) nanotubes were imaged 24 hours afterinternalization within HeLa cells; the nanotubes localized withinlysosomes in the perinuclear region. Subsequently, (8,6) nanotubes wereimaged immediately after a 30-minute incubation with the same cells,resulting in the localization of these nanotubes within endosomes only.The labeling thus spatially distinguished endosomes and lysosomes bytwo, narrow near-infrared spectral bands (FWHM ˜26 nm), separated by 210nm.

A DNA-encapsulated nanotube complex, undergoing a continuoussolvatochromic shifting response, spatially and temporally trackedendosomal maturation in live HeLa cells. Separated (8,6) nanotubesencapsulated by an ss(GT)₆ oligonucleotide, with a median length of 236nm, were incubated with HeLa cells at 0.25 mg/L for 30 minutes beforerinsing and replacing with fresh media. The wavelength distribution ofthe nanotube complex in endosomes, measured immediately after uptake,was centered at 1200.7±0.4 nm, near the bulk value observed in water. Inlysosomes, after 6 hours post-incubation, the wavelength distributionbroadened before blue-shifting to 1194.3±0.7 nm at the 24-hour timepoint(FIG. 2, panel A). A small red-shifted nanotube population, with a meanwavelength of 1205 nm, appeared at the 6-hour time point but disappearedby 24 hours. The relative mean wavelength of each emissive region ofinterest (ROI) was laid over a transmitted light image of the cells,resulting in spatial visualization of the spectral distribution (FIG. 2,panel B). Both the inter- and intra-cellular heterogeneity of emissionwavelengths progressively increased with endosomal maturation. Thenanotube wavelength-shifting phenomenon was found to differ among celltypes; MCF-10A epithelial cells caused blue-shifting similar to theobservation in HeLa cells, nanotubes in RAW 264.7 macrophages exhibitedlittle change relative to bulk nanotubes suspended in water, and GM05659human fibroblasts produced a red-shifted nanotube population. Theinteraction of GT₆-(8,6) nanotubes with RAW 264.7 macrophages wascharacterized, by incubating 2 pM of nanotubes in cell media for 30minutes at 37° C. Under these conditions, the majority of cellsefficiently took up nanotubes via energy-dependent mechanisms.

Example 4: Identifying a Carbon Nanotube Optical Reporter of LocalDielectric Enivronment

As nanotube photoluminescence exhibits blue-shifting behavior in lowerdielectric environments, the sensitivity of the ss(GT)₆oligonucleotide-encapsulated (8,6) nanotube complex to solvents withknown dielectric constants was directly tested. The complex wasincubated with butanol (ε=18), resulting in a 14.6±0.9 nm blue shiftfrom the corresponding emission in water (ε=80). In contrast,ss(TAT)₄-encapsulated, separated (6,5) nanotube complexes wererelatively less sensitive to the local dielectric environment (2.2±0.2nm blue shift in butanol) and reported negligible wavelength-shifting inlive HeLa cells, even at 24 hours. Therefore, the ss(GT)₆-(8,6) complexfunctions as a carbon nanotube optical reporter (CNOR) of localdielectric environment.

Pharmacological inhibition of endosomal maturation by the microtubulepolymerization inhibitor nocodazole prevented the blue-shifting of CNORemission in HeLa cells (FIG. 2, panels C-D). The maturation process ofendosomes containing the CNOR by adding nocodazole to the cell media at10 μg/mL 30 minutes prior to the incubation with the CNOR was halted;these CNORs did not blue-shift for up to 24 hours after internalization(1200.07±1.4 nm mean emission compared to 1194.28±0.73 for control).This result indicates that blue-shifting of the CNOR requires theendosome to lysosome transition and the corresponding change indielectric environment.

As the CNOR transitions from early endosomes to lysosomes, theenvironmental pH decreases from pH 6.6 to pH 4.5. To probe the effect ofpH on CNOR emission wavelength shifting, NH₄Cl—known to increaseendosomal pH—was used to alkalinize endocytic organelles in HeLa cells.On addition of 50 mM NH₄Cl to cells 6 hours after incubation with theCNOR, the blue-shifted population remained unaffected while thered-shifted population was depleted. This result, combined with theobserved red-shift of the CNOR with lowering pH suggests that thelowering of pH directly causes red-shifting of DNA-nanotube emission butis not responsible for the blue-shifting phenomenon.

The blue-shifting of CNOR emission was enhanced by the drug U18666A, anamphiphilic aminosteroid known to increase the cholesterol content oflate endosomes (FIG. 2, panels C-D). HeLa cells treated with 3 μg/mL ofthe drug for six hours before incubation with the CNOR resulted in afurther 3.8±0.7 nm increase in blue shift at 24 hours than in untreatedcontrol cells.

A similar effect was observed in RAW 264.7 macrophages; the CNORexhibited a blue-shift of 11.1±1.1 nm in drug-treated cells relative toa drug-free control. RAW macrophages were incubated in lipoproteindepleted serum (LPDS) for 24 hours. Incubation in these conditionsreduces the lipid content of the lysosomes and increases the number ofsurface receptors for acetylated LDL (Ac-LDL) on the cell surface. Toinduce lipid accumulation in the lysosomes, Ac-LDL and U18666A (acholesterol transport inhibitor) were introduced to cells incubating inthe lipoprotein depleted serum. After 3 hours of incubation, thecombination of Ac-LDL and U18666A results in a significant accumulationof unesterified cholesterol in the lysosomes of treated cells. Nanotubeswere added in normal cell media for 30 minutes, before free nanotubeswere washed away and the endocytosed nanotubes were given 3 hours tolocalize to the lysosomes. Hyperspectral images of the nanotubes withincells were acquired, and the images color coded to the peak emissionwavelength from each pixel. The hyperspectral images from nanotubes incells incubated in LPDS were mostly orange and red, while the cellstreated with Ac-LDL and U18666A appeared blue and green (FIG. 1, panelD). The histogram of the total nanotube emission from cells in eachcondition indicates that the nanotube emission from lipid-deficientlysosomes is ˜1200 nm while the emission from lipid-rich lysosomes is˜1192 nm (FIG. 1, panel E).

To understand why the accumulation of cholesterol within the constrainedvolume of a lysosome could induce a blue shift in the emission of theGT₆-(8,6) nanotube, all-atom replica exchange molecular dynamicssimulations were perfromed of the nanotube, and the nanotube withcholesterol in its vicinity. The equilibrium configurations obtainedafter 100 ns indicate that cholesterol partially self-assembles andbinds to exposed regions on the nanotube surface. In contrast to thestarting configuration, cholesterol also induces rearrangement of DNA onthe nanotube surface (FIG. 1, panel F). The net effect of cholesterolbinding and DNA rearrangement is a significant decrease in the densityof water molecules near nanotube surface (FIG. 1, panel G). Consistentwith the shift in nanotube emission due to environmental dielectric(FIG. 1, panel B), displacement of water from the nanotube surfaceresults in a relative blue shift.

Labeling unesterified cholesterol using filipin, which required fixingthe cells, displayed a marked qualitative difference between control anddrug-exposed cells. In control HeLa cells, filipin strongly illuminatedthe cholesterol-rich plasma membrane with only a small amount ofcholesterol-bound filipin emission appearing from within the cell, whilethe intensity of filipin in the cell interior increased greatly uponU18666A exposure (FIG. 2, panel D). These results indicate that the CNORemission undergoes blue-shifting in the presence of endosomal lipidsincluding free cholesterol, the major species up-regulated by the drug.

To confirm the ability of a water-soluble cholesterol derivative todirectly affect the emission of the nanotube reporter, the CNOR withsodium deoxycholate (SDC) was probed. The reporter, pre-incubated withcell culture media and serum, was adsorbed to a glass coverslip. A 0.1%solution of SDC was introduced, resulting in an average blue-shift of11.0±0.7 nm from 1201.5 nm to 1190.5 nm. The blue-shifted CNOR largelyreverted back to its initial emission wavelength upon removing SDC. Bysuccessively acquiring hyperspectral cubes, the solvatochromic responsewas found to reach equilibrium in less than 300 seconds. This experimentdemonstrates the intrinsic reversibility and rapid response of thereporter to changes in dielectric environment. It suggests that in acellular environment, the DNA-encapsulated nanotube leaves enoughexposed surface such that sterols or other amphipathic molecules canreversibly bind, resulting in an accumulative solvatochromic response tothe change in the dielectric environment of the CNOR.

The emission from other (n,m) species in an unsorted ss(GT)₆oligonucotide-encapsulated nanotube sample exhibited similar results tothe (8,6) nanotube, with varying degrees of blue-shift observed as afunction of time after uptake by HeLa cells. Changing the encapsulatingoligonucleotide sequence from ss(GT)₆ prevented the blue-shiftingphenomenon, indicating sequence-specificity of the optical response. Totest whether the CNOR remains intact within the lysosome,ss(GT)₆-encapsulated nanotubes was labeled with Cy3 and the organicfluorophore was found to remain quenched by the nanotube surface. Thisfinding confirms the stability of the sensor, as the encapsulating DNAremains associated with the nanotube.

Example 5: Identifying Lysosomal Storage Disorders

Niemann Pick Type-C (NPC) is a lysosomal storage disorder characterizedby massive accumulation of unesterified cholesterol in the lysosomes ofpatient fibroblasts. The solvatochromic shifting of nanotubes clearlydistinguished wild-type (WT) fibroblasts from fibroblasts acquired froma patient with NPC. The NPC cells accumulate cholesterol and otherlipids within their late endosomes and lysosomes due to a homozygousmutation of the NPCI gene. At 24 hours after the initial 30 minuteincubation with the CNOR, its emission blue-shifted by approximately 10nm in NPC cells compared to WT fibroblasts (1203.1±0,48 nm for NPC vs.1209.9±0.32 nm for WT, FIG. 3, panel A). The solvatochromic response wasmonitored spatially across the cells, showing variations among endosomalcompartments (FIG. 3, panel B), but exhibiting clear, quantitativedifferences between diseased and non-diseased cells (p-value<0.005forspectral identification of NPC and WT cells).

Treatment of homozygous NPC cells with cyclodextrin (HPβCD), a moleculewhich removes free cholesterol from the lumen of the lateendosome/lysosome, caused a reversal of the CNOR blue-shift, thusbenchmarking the therapeutic action in live cells. Subsequent to imagingat 24 hours after incubation with nanotubes, NPC fibroblasts wereincubated with HPβCD for an additional 24 hours (histograms and overlaysin FIG. 3, panels C-D). The effect of HPβCD treatment on CNOR emissionfrom NPC cells was dramatic; the blue-shifted nanotube populationtransitioned to a red-shifted population and appeared nearly identicalto nanotubes in WT cells (FIG. 3, panel E). This result correlated wellwith staining by filipin, indicating that cyclodextrin removed freecholesterol from the lumen of the late endosome/lysosome, likely drivingcholesterol off the nanotube surface and thereby reversing itsblue-shifted emission state. Pre-treatment of NPC cells with HPβCDprevented CNOR fluorescence from shifting in diseased cells, resultingin emission that was spectrally identical (p<0.005) to that in WTfibroblasts. Thus, the excess free cholesterol in the lateendosome/lysosome of NPC cells is responsible for the blue shiftobserved when compared to healthy fibroblasts, and removal of the excessfree cholesterol inhibits the blue-shift of the CNOR.

The blue-shifting phenomenon was also demonstrated on fibroblasts from adifferent patient, with the compound heterozygous mutations of the NPC1gene, which also exhibit substantial cholesterol accumulation. Thelysosomes in NPC1 cells were heterogeneous in their lipid content, incontrast to WT fibroblasts. The reporter was incubated for 30 minuteswith cells acquired from a 10-year old patient, before free nanotubeswere washed away. Measurements at 24 hours indicated nanotubes in thelysosomes of NPC1 cells to be blue shifted by 6 nm, compared with WTfibroblasts (FIG. 4, panel A). To test the ability of the reporter todetect disease reversal, the NPC1 cells were treated with 100 uMcyclodextrin for 24 hours. This significantly red-shifted the nanotubeemisson by approximately 4 nm—and indicated that within the lysosomes oflive cells, the reporter is reversible and accurately detected thedecrease in cholesterol due to the therapeutic effect of cyclodextrin.By looking at histograms obtained from single lysosomes from cells at 48hours, WT fibroblast lysosomes were found to be uniformly lipid-poor,with a homogeneous distribution centered at ˜1210 nm (FIG. 4, panel B).In contrast, the lysosomes from NPC1 cells showed a broad distribution,and included a lipid-poor fraction, indicating that not all lysosomesdemonstrate lipid accumulation. The cyclodextrin treated NPC cellsshowed a narrow homogeneous distribution of lysosomal lipid content,similar to the distribution observed in WT fibroblasts.

The nanotube results were confirmed by staining the cells with filipin,a fluorescent fixed cell marker with high specificity for cholesterol.Though filipin is not organelle specific, cholesterol accumulation inNPC1 is limited to the lysosomes and thus increased filipin intensityindicates an increase in the cholesterol content of affected lysosomes.Filipin staining confirmed the nanotube emission data as being accurate,with higher filipin intensity observed in blue-shifted cells (FIG. 4,panel C). To confirm that the reporter was specifically detectingcholesterol, NPC1 cells were pre-treated with cyclodextrin. Aftertreatment, the lipid content of these lysosomes should approximate WTcells. Consistent with this finding, nanotubes added to cyclodextrinpretreated cells, and to WT fibroblasts displayed identical emissionvalues, which were significantly red-shifted from untreated NPC1fibroblasts (FIG. 4, panel D).

The reporter detected Wolman's disease, a lysosomal storage disordercharacterized by the accumulation of esterified cholesterol in thelysosomes due to a non-functioning lysosomal acid lipase (LAL).Lalistat, a specific inhibitor of LAL, was used to prevent thehydrolysis of esterified cholesterol. As a positive control in RAWmacrophages, the NPC1 phenotype was induced by preventing cholesterolefflux from lysosomes with U18666A. Compared with the controlmacrophages, both U18666A and Lalistat induced a 8 nm blue shift (FIG.4, panel E). Therefore, the reporter blue-shifts due to an accumulationof lipidic molecules in the lysosomes of cells. The lysosomalaccumulation of unesterfied cholesterol in NPC1 or esterifiedcholesterol in Wolman's dieases both result in the binding of lipidmolecules to the nanotube surface and induce a blue shift in thenanotube emission (FIG. 4, panel G).

Cells can accumulate lipids within their lysosomes at different rates,and the reporter was used to understand the parameters of lipidaccumulation in individual cells. The reporter was localized in thelysosomes of RAW macrophages incubated in lipoprotein depleted serum.The emission from nanotubes in these lipid-poor lysosomes was at 1200nm. Upon adding both AcLDL and U18666A to the media, hyperspectralimages of the same cells were acquired every 10 minutes for a two hourperiod. Cells progressively blue shifted due to unesterified cholesterolaccumulation within their lysosomes (FIG. 5, panel A). Single cellsacross hyperspectral images were tracked and the mean nanotube emissionfrom each cell for every time point was extracted. The rate of blueshifting of individual cells can be described as a lag period of noshifting, followed by a single exponential decay. Each singleexponential decay can be fit to obtain the time constant for each cell(FIG. 5, panel B) - the average time constant of lipid accumulation is40 minutes. The distribution of time constants from individual cellsfollows a log normal distribution, which is consistent with the rate oflipid accumulation within the lysosomes of a single cell being aconvolution of multiple independent processes (FIG. 5, panel C). Cellsshow remarkable heterogeneity in their rate of lipid accumulation, withthe slowest and fastest time constants differing by an order ofmagnitude.

The slowest cells were found to be the ones that started with the mostlipid-deficient lysosomes. The time constant of lipid accumulationcorrelated with the starting wavelength (Spearman correlation of 0.33,p<0.01), but interestingly, all slow cells (defined as cells with a timeconstant greater than 90 minutes) started off with an emission greaterthan 1202 nm. The time lag before the single exponential blue shiftingcommenced did not correlate to the starting emission value, the finalemission value, the total emission shift or the time constant of decayfor single cells. Therefore, cells accumulate lipids in the lysosomes ata rate independent of a lag period preceding lipid accumulation. Therate of lipid accumulation is heterogeneous, and cells slow toaccumulate lipids within their lysosomes also start with the mostlipid-deficient lysosomes. Lipid accumulation due to U18666A action inthe presence of AcLDL uptake is a complex process. Using the reporterdirectly measures the phenomenon of interest, lysosomal lipidaccumulation.

Example 6: Identifying Differentiation State of Bone Marrow

The reporter can correlate lysosomal lipid content with thedifferentiation state of bone marrow derived macrophages (BMDM).Monocytes in the presence of colony-stimulating factor (CSF1)differentiate into macrophages, as observed by an increase in thefraction of cells expressing the macrophage markers CD11b and F4/80 anda decrease in the fraction of cells expressing the monocyte marker GR1(FIG. 6, panel A). Nanotubes taken up by BMDM under standardexperimental conditions localize in the lysosomes and appear as punctatespots under 100× magnification (FIG. 6, panel B). Each emissive pixel isfitted to obtain the nanotube emission peak, and thus generates alysosomal lipid map of individual cells (FIG. 6, panel C). The averagenanotube emission from each pixel within a cell gives a mean lysosomallipid value for that particular cell. A collection of these meannanotube emission values thus benchmarks a population of cells by thedegree of lipid accumulation within their lysosomes. As macrophagesdifferentiate from day 3 to day 5, the nanotube emission significantlyblue shifts by ˜5 nm (FIG. 6, panel D), indicating that day 5macrophages have significantly greater lysosome lipid content than day 3macrophages.

The process of lysosomal lipid accumulation was observed within singlecells, by quantifying the intra cellular heterogeneity in lysosomallipid content. The nanotube emission from lysosomes within a cell wasanalyzed with two parameters - an average emission value, representingthe overall state of lysosomal lipid accumulation within that cell, anda normalized Simpson's Index, to quantify the degree of heterogeneitybetween lysosomes within that cell. Two typical cells from day 3,showing a relatively homogeneous red cell with a normalized Simpson'sIndex (nSI) value of 0.18, and a more heterogeneous blue cell with a nSIof 0.74 are shown (FIG. 6, panel E). Plotting the mean emission value ofa cell against its nSI demonstrates a clear partitioning, as all bluecells are highly heterogeneous (FIG. 6, panel F). Blue cells being themost heterogeneous indicates that as cells mature and transition fromred (lipid deficient lysosomes) to blue (lipid rich lysosomes),individual lysosomes within a single cell also transition from red toblue, which results in an increase in the heterogeneity (both red andblue lysosomes) observed within single cells. The application of thereporter to study differentiating macrophages informs us that lysosomallipid content increases as monocytes differentiate into macrophages, andthis process is observable at the single cell level.

In summary, the above-described non-photobleaching fluorescent probe,localizes to the lysosome, and reports variations in the lipid contentof individual lysosomes within live cells. Spatially localizing thenanotube emission generates a endolysosomal lipid map of a cell, andchanges in the lysosomal lipid content can be measured over prolongedperiods of time with single cell and single lysosome resolution. Thiscarbon nanotube optical reporter indicates that changes in the lysosomallipid content can occur both due to dysfunctions such as lysosomalstorage disorders and normal cellular processes such as macrophagedifferentiation.

1. A method of detecting a condition in a subject using single-walledcarbon nanotubes (SWCNTs), comprising: contacting cells of said subjectwith SWCNTs; monitoring photoluminescence emitted by SWCNTs internalizedinto said cells and generating an SWCNT emission profile; comparing theSWCNT emission profile to a control emission profile for the SWCNTs toproduce a result; and determining a likelihood of having said conditionin said subject based on the result from said comparing step. 2.(canceled)
 3. (canceled)
 4. The method of claim 1, wherein the SWCNTsare semi-conductive SWCNTs.
 5. The method of claim 1, wherein the SWCNTsare oligonucleotide-coated non-covalently encapsulated SWCNTs.
 6. Themethod of claim 1, wherein the SWCNTs are encapsulated by DNAoligonucleotides, optionally the DNA oligonucleotides are ss(GT)₆oligonucleotides.
 7. The method of claim 1, wherein the SWCNTs havechiral indices (n,m) of (6,5) or (8,6).
 8. The method of claim 1,wherein the SWCNTs are purified (8,6) SWCNTs encapsulated with ss(GT)₆oligonucleotides.
 9. The method of claim 1, wherein the condition is adisease linked to elevated lipid and cholesterol content and wherein ablue-shift in the SWCNT emission profile from the control emissionprofile is an indication of the present of the condition in the subject.10. The method of claim 9, wherein the condition is atherosclerosis,fatty liver disorder or cancer.
 11. The method of claim 9, wherein thecondition is hypercholesterolemia.
 12. The method of claim 1, whereinthe cells contacted with SWCNTs are cultured in vitro.
 13. The method ofclaim 1, wherein the cells contacted with SWCNTs are fibroblasts,microphages or hepatocytes.
 14. The method of claim 1, wherein the cellscontacted with SWCNTs are located in a subject in vivo.
 15. The methodof claim 1, further comprising the step of: extracting cells from thesubject; and culturing the extracted cells in vitro to produce culturecells, wherein the cells contacted with SWCNTs are the cultured cellsfrom the culturing step.
 16. The method of claim 3, wherein the sampleis a biological sample.
 17. The method of claim 16, wherein the sampleis a plasma sample. 18-22. (canceled)
 23. A kit for detecting changes inlipid accumulation, comprising: SWCNTs; and oligonucleotides suitablefor encapsulating SWCNTs.
 24. The kit of claim 23, wherein the SWCNTsare (8,6) carbon nanotubes and the oligonucleotides are ss(GT)₆oligonucleotides.